This study investigated the effect of vomitoxin (DON) on intestinal motility in 6-week-old male C57BL/6J mice. Sixty mice were randomized into control and DON-treated groups, with six replicates of five mice each. The control group was gavaged with saline and the DON-treated group was gavaged with 3 mg/kg body weight of DON for 14 days. The results showed that DON significantly decreased the feed intake and weight gain (P＜0.001) and inhibited the growth of mice; shortened the intestinal propulsive distance, decreased the villus height, villus height/crypt depth, and intestinal wall thickness (P＜0.001), and disrupted the intestinal morphology and structure; down-regulated the expression of intestinal motility-related genes and proteins (P＜0.001); increased the number of M1-type inflammatory macrophages and activated the TLR-MAPK signaling pathway, up-regulated pro-inflammatory cytokine expression, and down-regulated anti-inflammatory cytokine expression (P＜0.001); activated the TNFR1-Casp3 signaling pathway and increased the secretion of apoptotic marker proteins (P＜0.001); and decreased the number of ICC cells (P＜0.001). This study demonstrated that DON causes intestinal motility disorders and morphological and structural damage and is associated with changes in the expression of related gene proteins, activation of inflammatory signaling pathways, activation of pro-apoptotic signaling pathways, and a decrease in the number of ICC cells. This study provides a theoretical basis for understanding the mechanism of DON toxicity and developing prevention and control strategies.